The present invention is directed to a series of ligands, and more particularly to estrogen receptor-xcex2 ligands which have better selectivity than estrogen for the estrogen receptor-xcex2 over the estrogen receptor-xcex1, as well as to methods for their production and use in the treatment of diseases related to the estrogen receptor-xcex2, specifically. Alzheimer""s disease, anxiety disorders, depressive disorders, osteoporosis, cardiovascular disease, rheumatoid arthritis, or prostate cancer.
Estrogen-replacement therapy (xe2x80x9cERTxe2x80x9d) reduces the incidence of Alzheimer""s disease and improves cognitive function in Alzheimer""s disease patients (Nikolov et al. Drugs of Today, 34(11), 927-933 (1998)). ERT also exhibits beneficial effects in osteoporosis and cardiovascular disease, and may have anxiolytic and anti-depressant therapeutic properties. However, ERT shows detrimental uterine and breast side effects that limit its use.
The beneficial effects of ERT in post-menopausal human women is echoed by beneficial effects of estrogen in models relevant to cognitive function, anxiety, depression, bone loss, and cardiovascular damage in ovariectomized rats. Estrogen also produces uterine and breast hypertrophy in animal models reminiscent of its mitogenic effects on these tissues in humans.
The beneficial effects of ERT in post-menopausal human women is echoed by beneficial effects of estrogen in models relevant to cognitive function, anxiety, depression, bone loss, and cardiovascular damage in ovariectomized rats. Specifically, experimental studies have demonstrated that estrogen effects the central nervous system (xe2x80x9cCNSxe2x80x9d) by increasing cholinergic function, increasing neurotrophin/neurotrophin receptor expression, altering amyloid precursor protein processing, providing neuroprotection against a variety of insults, and increasing glutamatergic synaptic transmission, among other effects. The overall CNS profile of estrogen effects in pre-clinical studies is consistent with its clinical utility in improving cognitive function and delaying Alzheimer""s disease progression. Estrogen also produces mitogenic effects in uterine and breast tissue indicative of its detrimental side effects on these tissues in humans.
The estrogen receptor (xe2x80x9cERxe2x80x9d) in humans, rats, and mice exists as two subtypes. ER-xcex1 and ER-xcex2, which share about a 50% identity in the ligand-binding domain (Kuiper et al. Endocrinology 139(10) 4252-4263 (1998)). The difference in the identity of the subtypes accounts for the fact that some small compounds have been shown to bind preferentially to one subtype over the other (Kuiper et al.).
In rats. ER-xcex2 is strongly expressed in brain, bone and vascular epithelium, but weakly expressed in uterus and breast, relative to ER-xcex1. Furthermore, ER-xcex1 knockout (ERKO-xcex1) mice are sterile and exhibit little or no evidence of hormone responsiveness of reproductive tissues. In contrast, ER-xcex2 knockout (ERKO-xcex2) mice are fertile, and exhibit normal development and function of breast and uterine tissue. These observations suggest that selectively targeting ER-xcex2 over ER-xcex1 could confer beneficial effects in several important human diseases, such as Alzheimer""s disease, anxiety disorders, depressive disorders, osteoporosis, and cardiovascular disease without the liability of reproductive system side effects. Selective effects on ER-xcex2-expressing tissues (CNS, bone, etc.) over uterus and breast could be achieved by agents that selectively interact with ER-xcex2 over ER-xcex1.
It is a purpose of this invention to identify ER-xcex2-selective ligands that are useful in treating diseases in which ERT has therapeutic benefits.
It is another purpose of this invention to identify ER-xcex2-selective ligands that mimic the beneficial effects of ERT on brain, bone and cardiovascular function.
It is another purpose of this invention to identify ER-xcex2-selective ligands that increase cognitive function and delay Alzheimer""s disease progression.
This present invention is directed to the use of compounds having the generic structure: 
as ER-xcex2-selective ligands, which mimic ERT, but lack undesirable side effects of ERT. These compounds particularly satisfy the formula:
(Kixcex1A/Kixcex2A)/(Kixcex1E/Kixcex2E) greater than 1,
preferably:
(Kixcex1A/Kixcex2A)/(Kixcex1E/Kixcex2E) greater than 30,
more preferably:
(Kixcex1A/Kixcex2A)(Kixcex1E/Kixcex2E) greater than 100,
wherein Kixcex1A is the Ki value for the ligand in ER-xcex1; Kixcex2A is the Ki value for the ligand in ER-xcex1: Kixcex1E is the Ki value for estrogen in ER-xcex1; and Kixcex2E is the Ki value for estrogen in ER-xcex2.
The instant invention involves a method for treating a disease associated with the estrogen receptor-xcex2, comprising the step of administering a therapeutically-effective amount of a compound that satisfies the equation (Kixcex1A/Kixcex2A)/(Kixcex1E/Kixcex2E) greater than 1, wherein Kixcex1A is the Ki value for the agonist in ER-xcex1; Kixcex2A is the Ki value for the agonist in ER-xcex2; Kixcex1E is the Ki value for estrogen in ER-xcex1; and Kixcex2E is the Ki value for estrogen in ER-xcex2. Preferably, the compound satisfies the equation (Kixcex1A/Kixcex1A)/(Kixcex1E/Kixcex2E) greater than 100. Preferred diseases associated with the estrogen receptors xcex2 are selected from Alzheimer""s disease, anxiety disorders, depressive disorders, osteoporosis, cardiovascular disease, rheumatoid arthritis and prostate cancer. More preferably, the diseases are Alzheimer""s disease or depressive disorders.
The compounds of the instant invention are ER-xcex2-selective ligands of the structure: 
In this structure L1 is xe2x80x94C(xe2x95x90O)xe2x80x94, xe2x95x90C(R6)xe2x80x94, xe2x80x94CH(R6)xe2x80x94, O, S, or NRa, preferably xe2x80x94C(xe2x95x90O)xe2x80x94, xe2x95x90C(R6)xe2x80x94, xe2x80x94CH(R6)xe2x80x94or O; L2 is xe2x95x90Cxe2x80x94or xe2x80x94CHxe2x80x94; L3 is xe2x95x90C(R6)xe2x80x94, xe2x80x94CH(R6)xe2x80x94or xe2x80x94C(xe2x95x90O)xe2x80x94; and L4 is xe2x80x94C(xe2x95x90O)xe2x80x94, CH2, O, S, or NRa, preferably xe2x80x94C(xe2x95x90O)xe2x80x94, CH2, or O, provided that when L1 is xe2x80x94C(xe2x95x90O)xe2x80x94; L4 is CH2, O, S, or NRa; when L4 is xe2x80x94C(xe2x95x90O)xe2x80x94, L1 is CH2, O, S, or NRa; and when L3 is xe2x80x94C(xe2x95x90O)xe2x80x94, L1 is xe2x95x90C(R6)xe2x80x94or xe2x80x94CH(R6)xe2x80x94, and L4 is O or NRa. Additionally, when L1 is xe2x95x90C(R6)xe2x80x94, L2 is xe2x95x90Cxe2x80x94; when L1 is xe2x80x94CH(R6)xe2x80x94, L2 is xe2x80x94CHxe2x80x94; when L3 is xe2x95x90C(R6)xe2x80x94, L2 is xe2x95x90Cxe2x80x94; and when L3 is xe2x80x94CH(R6)xe2x80x94. L2 is xe2x80x94CHxe2x80x94. xe2x95x90 represents a single bond or double bond, depending upon the hybridization of L1-L4. The structures for L2 show only three bonds because the fourth bond is a single bond to R1.
R1 is attached via a single bond to L2, and is phenyl, substituted phenyl, Het, or substituted Het, as defined below. R1 is preferably: 
wherein: R7 is H, Cl, or methyl; R8 is Br, Cl, F, Ra, ORa, or allyl; R9 is H, OH, NH2, Br, Cl; and R10 is H or methyl: or R8 and R9 may combine to be xe2x80x94OCH2Oxe2x80x94, forming a secondary 5-membered ring structure exterior to the phenyl group; or R1 is a substituted or unsubstituted heterocyclic substituent having the following structure: 
more preferably unsubstituted 
R2, R3, R4, and R5 are each independently, xe2x80x94Ra, xe2x80x94ORa, xe2x80x94SRa, xe2x80x94NRaRa, xe2x80x94NC(xe2x95x90O)Ra, xe2x80x94NS(xe2x95x90O)Ra, xe2x80x94NS(xe2x95x90O)2R2, halogen, cyano. xe2x80x94CF3, xe2x80x94CO2Ra, xe2x80x94C(xe2x95x90O)Ra, xe2x80x94C(xe2x95x90O)NHRa, nitro, xe2x80x94S(xe2x95x90O)Ra, or xe2x80x94S(xe2x95x90O),Ra, and is preferably Ra, ORa, NR2a, NC(xe2x95x90O)Ra, CF3, or halogen, preferably, hydrogen, hydroxyl or methyl.
R6 is Ra, phenyl or CF3.
Ra is, independently, at each occurrence, H or (C1-C5)alkyl.
When L1 is xe2x80x94C(xe2x95x90O)xe2x80x94, and R2 is hydroxy or hydrogen, and R3 is hydrogen, and R4 is hydroxy, and R5 is hydrogen, and R6 is hydrogen, then R1 is not para-phenol.
For purposes of this invention, xe2x80x9csubstitutedxe2x80x9d when used to modify a phenyl or a heteroatomic ring means such a ring substituted at one or more positions, independently, with xe2x80x94Ra, xe2x80x94ORa, xe2x80x94SRa, xe2x80x94NRa, Ra, xe2x80x94NC(xe2x95x90O)Ra, xe2x80x94NS(xe2x95x90O)Ra, xe2x80x94NS(xe2x95x90O)2, Ra, halogen, cyano, xe2x80x94CF3, xe2x80x94CO2Ra, xe2x80x94C(xe2x95x90O)Ra, xe2x80x94C(xe2x95x90O)NHRa, nitro, xe2x80x94S(xe2x95x90O)Ra, or xe2x80x94S(xe2x95x90O)2Ra.
Also, for purposes of this invention, xe2x80x9cHetxe2x80x9d means a substituted or unsubstituted one- or two-ring heterocycle selected from the following: 
wherein the crossed bond represents that the heterocycle may be attached at any available position on the ring that it crosses.
The ability of a compound to bind to ER was measured by its ability to compete for binding with the radio-labeled estrogen. [125I]-16xcex1-iodo-3,17xcex2-estradiol (NEN, Cat.#NEX-144). The radio-ligand is hereafter referred to as [125I]-estradiol.
ER-xcex2 (Gen Bank Accession #X99101) or ER-xcex1 (Gen Bank Accession #M12674) cDNAs were cloned into the expression vector pSG5 (Stratagene), transformed into e. coli strain DHxcex1Fxe2x80x2, and purified using anion-exchange resin columns (Qiagen Cat.#12125). Receptor protein was prepared by in vitro transcription and translation of these plasmids using the TNT T7 Quick-Coupled reticulocyte lysate system (Promega Cat.#L1170). Reticulocyte lysate (12.5 mL) was incubated for 90 min at 30xc2x0 C. with 312.5 xcexcg of ER-xcex1 and 625 xcexcg of ER-xcex2 plasmids. Programmed lysate was then aliquotted and stored frozen at xe2x88x9280xc2x0 C.
Compounds were tested in duplicate at half-log concentrations ranging from 10 pM to 13 xcexcM. Compounds were prepared as 1 mM stocks in DMSO, then diluted in the binding-assay buffer (in mM: 20 HEPES, 150 NaCl, 1 EDTA, 6 monothioglycerol and 10 Na2MoO4; 10% wt/vol glycerol, and pH =7.9) to a series of three-fold concentrated, 20 xcexcL aliquots in a 96-well plate. Receptor aliquots were thawed on ice, and appropriately diluted (see below) in binding assay buffer. Diluted receptor (30 xcexcL/each) was added to each well. [125I]-estradiol was diluted from the manufacturer""s ethanol stock solution to a 900 pM working solution in binding-assay buffer. The final assay volume was 60 xcexcL, consisting of 20 xcexcL of a compound according to the instant invention, 30 xcexcL of programmed reticulocyte lysate, and 10 xcexcL of 900 pM [125I]-estradiol. The final concentration of [125I]-estradiol was 150 pM. Plates containing the final assay mixture were mixed on a shaker for 2 min and incubated overnight (xcx9c16 h) at 4xc2x0 C.
Receptor-bound and unbound radioligand was separated by filtration over sephadex columns. Columns (45 xcexcL bed volume) were prepared by adding dry column media (Pharmacia Cat#G-25) to 96-well column templates (Millipore MultiScreen Plates Cat#MAHVN4510). Columns were then saturated with 300 xcexcL of binding-assay buffer and stored at 4xc2x0 C. Prior to use, stored columns were spun for 10 minutes at 2000 RPM, then washed twice with 200 xcexcL of fresh binding buffer. The binding-assay mixtures (50 xcexcL/each) were then applied to the columns, and an additional elution volume of 35 xcexcL was immediately applied to the column. Receptor-bound radioligand was then cluted from the column by centrifugation for 10 minutes at 2000 RPM. A scintillation cocktail (145 xcexcL) was added to the eluted radioligand/receptor complex, and radio-label was measured by liquid scintillation counting.
Non-specific binding was defined by competition with 150 nM diethylstilbesterol (DES) Binding affinities are expressed as Ki, calculated using the Cheng-Prushoff formula according to IC50 values generated by fitting the relationship of concentration to percent specific binding (SB) with the following equation:
% SB=Maximumxe2x88x92(Maximumxe2x88x92Minimum)/(1+10(logIC50-log[compound])) In this assay, standard estrogen receptor ligands estradiol and DES were detected as high-affinity (Ki less than 1 nM), non-selective ligands of ER-xcex2 and ER-xcex1.
The volume of receptor-programmed reticulocyte lysate to be added to the binding assay was determined independently from two measurements made on each batch of receptor prepared. First, Kis were determined for standard compounds using a series of dilutions of the receptor preparation. Scatchard analysis of ligand binding affinity was performed at the receptor dilutions that produced reported Kis for these compounds and an acceptable signal:noise ratio (xcx9c10). These experiments indicated a KD for [125I]-estradiol of 0.1-1 nM, and a Bmax of 5-30 pmol.
Compounds of the present invention are shown to have high selectivity for ER-xcex2 over ER-xcex1, and may possess agonist activity on ER-xcex2 without undesired uterine effects. Thus, these compounds, and compositions containing them, may be used as therapeutic agents in the treatment of various CNS diseases related to ER-xcex2, such as, for example. Alzheimer""s disease.
The present invention also provides compositions comprising an effective amount of compounds of the present invention, including the nontoxic addition salts, amides and esters thereof, which may, serve to provide the above-recited therapeutic benefits. Such compositions may also be provided together with physiologically-tolerable liquid, gel or solid diluents, adjuvants and excipients. The compounds of the present invention may also be combined with other compounds known to be used as therapeutic agents for the above or other indications.
These compounds and compositions may be administered by qualified health care professionals to humans in a manner similar to other therapeutic agents and, additionally, to other mammals for veterinary use, such as with domestic animals. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified. The active ingredient is often mixed with diluents or excipients which are physiologically tolerable and compatible with the active ingredient. Suitable diluents and excipients are, for example, water, saline, dextrose, glycerol, or the like, and combinations thereof. In addition, if desired the compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents stabilizing or pH-buffering agents, and the like.
The compositions are conventionally administered parenterally, by injection, for example, either subcutaneously or intravenously. Additional formulations which are suitable for other modes of administration include suppositories, intranasal aerosols, and, in some cases, oral formulations. For suppositories, traditional binders and excipients may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, or powders.
The present compounds may be formulated into the compositions as neutral or salt forms. Pharmaceutically-acceptable nontoxic salts include the acid addition salts (formed with the free amino groups) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
In addition to the compounds of the present invention that display ER-xcex2 activity, compounds of the present invention can also be employed as intermediates in the synthesis of such useful compounds.
Compounds within the scope of the present invention may be synthesized chemically by means well known in the art. The following Examples are meant to show general synthetic schemes, which may be used to produce many different variations by employing various commercially-available starting materials. These Examples are meant only as guides on how to make some compounds within the scope of the invention, and should not be interpreted as limiting the scope of the invention.